Field of the Invention
The present invention relates to polypeptides having phospholipase A activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.
Description of the Related Art
Phospholipases have been isolated from a wide number of organisms such as animals, plants, bacteria and fungi.
A lipase from Aspergillus niger (UNIPROT: B8YIE6) has an amino acid sequence that is 70% identical to the phospholipase of the present invention.
A phospholipase having A1 activity has been made available as LECITASE ULTRA by Novozymes A/S (Yang et al. Food Technol. Biotechnol. 44 (1) 101-104 (2006)).
A number of uses of phospholipases are known in the art, such as to use phospholipase in, e.g. enzymatic degumming of vegetable oil (Yang et al., Supra; U.S. Pat. No. 5,264,367, Metallgesellschaft, Röhm); treatment of starch hydrolysate (particularly from wheat starch) to improve the filterability (EP 219,269, CPC International); as an additive to bread dough to improve the properties of the dough and bread (U.S. Pat. No. 4,567,046, Kyowa Hakko); and for preparation of lyso-lecithin with special emulsifying properties.
Phospholipases may be applied for degumming of vegetable oils to provide refined storage stable vegetable oils of neutral taste and light color suitable for consumption. The degumming process comprises removing the phospholipid compounds also known as phosphatides or “the gum” from the triglyceride rich oil fraction.
Traditionally, the degumming process has been based on water extraction, with acidic or caustic treatment followed by a separation process. Due to the emulsifying properties of the phosphatides, the degumming procedure has resulted in a loss of oil i.e., of triglycerides. However, lately enzymatic degumming has become more widespread. Enzymatic degumming is performed on oils which have been water degummed as well as crude oils. In water degumming of edible oils, a part of the phosphatides is left in the oil. That part is described by the generic term “non-hydratable phosphatides” (NHP). In the production of oils, it is essential to remove the NHP content (U.S. Pat. No. 5,264,367). In the enzymatic degumming process, the NHP are converted by the use of phospholipase into water soluble and water extractable components.
The most widely applied commercial enzyme for industrial degumming of vegetable oils is the phospholipase LECITASE ULTRA from Novozymes A/S (Yang et al., Supra). LECITASE ULTRA has a relatively high thermostability; however, it is most suitable for use in degumming processes at temperatures of up to no more than 55° C.
Enzymatic degumming is generally assisted by the use of acid and caustic chemicals. In the initial degumming step addition of citric acid or phosphoric acid improves hydratability of salt forms of phospholipids by chelating the metals to citrate salts. In the following enzymatic reaction the pH often needs to be partly neutralized (typically by sodium hydroxide addition) to accommodate the pH requirements of the applied enzyme. When using the phospholipase LECITASE ULTRA optimum pH for this reaction is between 5.0 and 5.5 (see application sheet “Refining of vegetable oils—Improving degumming yield”). However, in this pH range, calcium and/or magnesium released during the enzymatic reaction combine with the buffering or chelating acids used to control the reaction conditions, typically leading to salt formation and fouling of equipment with deposits of calcium and magnesium citrate salts (U.S. Pat. No. 7,713,727). It would thus be advantageous if the enzymatic reaction could be performed at a lower pH reducing the need for neutralization of the acid applied.
There is an ever existing need for providing novel phospholipases with improved properties, such as increased activity at high temperature and/or low pH. The present invention relates to such novel polypeptides having phospholipase A activity and polynucleotides encoding the polypeptides.